Influence of the Host Plant on the Encyrtid Aenasius bambawalei , a Parasitoid used to Control the Cotton Mealybug , Phenacoccus solenopsis , in Pakistan

Hayat Badshah1, Farman Ullah1, Paul-André Calatayud2, Hidayat Ullah3 and Bashir Ahmad1 1Plant Physiology/Entomology Section, Agricultural Research Institute, Tarnab, Peshawar 2Institute of Research and Development c/o icipe African Insect Science for Food and Health, Noctuid Stem Borer Biodiversity Team, P.O. Box 30772-00100 Nairobi, Kenya 3Department of Agriculture, University of Swabi, Swabi, Pakistan


INTRODUCTION
T he mealybug Phenacoccus solenopsis (Sternorrhyncha: Pseudococcidae) is a highly polyphagous pest (Rashid et al., 2012), feeding on more than 154 plant species including fruits, vegetables, crops and ornamentals in Pakistan (Arif et al., 2009).Biological control approach by using Aenasius bambawalei (Hymenoptera: Encyrtidae) has been developed recently to control this pest (Arif et al., 2012;Fand et al., 2011;Venilla et al., 2010;Nagrare et al., 2009).However, the parasitism efficiency of this parasitoid is variable (Iqbal et al., 2016) according to the locality and the plants on which P. solenopsis is feeding (Arif et al., 2012;Persad et al., 2012).There is therefore a need to investigate the influence of the host plant of P. solenopsis on the efficiency of A. bambawalei.
Hymenopteran parasitoids lay their eggs in the body/ abdomen of their hosts and the parasitoid larvae are entirely dependent on the host for their development, as they feed internally in the host to complete their whole development (Godfray, 1995).Their fate is therefore strongly linked with the fate of the host, at an individual as well as at population level (Tanja et al., 2003).
Plants not only influence the herbivorous insect developments but also those of their associated natural enemies (Arif et al., 2012).Host plant affect parasitoid fitness by a variety of means, including survivorship, clutch size, body size, and/or fecundity and negative impacts on parasitoid fitness may occur when the developing parasitoid encounters the plant toxin in the hemolymph or tissue of its herbivorous host (Ode, 2006).Both physical and chemical characteristics of plants are known to influence significantly the efficacy of biological control agents by influencing their abundance, survival, development time, fecundity, and rate of attack (Consuelo et al., 2000).Host plants structure and morphology can strongly affect the location and acceptance ability of the parasitoids to exploit their hosts (Wang et al., 2009(Wang et al., , 2013)).Yang and Sadof (1995) reported that Coleus sp leaf variegation affects the rate of fecundity, survivorship, and population growth of both the citrus mealybug, Planococcus citri (Risso), and its parasitoid, Leptomastix dactylopii (Howard).Host plant quality or size is commonly correlated to parasitoid fitness, especially in idiobiont parasitoids, whose offspring may depend on the quality and size of the host at the time of oviposition (Gols and Harvey, 2008;Fortuna et al., 2012).
It has been well documented that the parasitism level of A. bambawalei varies with respect to the host plants (Arif et al., 2012;Fand et al., 2011;Venilla et al., 2010, Nagrare et al., 2009).However, the direct influence of the host plant on this parasitoid species was never demonstrated.In this context, we investigated the direct influence of the host plant, frequently found infested in the field by P. solenopsis, on the parasitism (behavioural aspects, success of parasitism and fitness) of A. bambawalei under field and laboratory conditions through choice and no choice bioassays.

Plants
Five plant species (hibiscus, potato, okra, eggplant and tomato) were used in this study because P. solenopsis is frequently found to feed on them in the field (Arif et al., 2009).For field experiments, plants of hibiscus of a minimum of 3 years old grown in the Institute as ornamental plants were directly used.Plants of eggplant and tomato were grown first at a nursery in the field during 4-6 weeks before to be transplanted individually in small pots while plants of potato and okra were directly raised in the small pots during 4-6 weeks for experiments.For laboratory experiments, the shoots (12-15 cm) with tender leaves were collected from each of the four plants and fixed in glass conical flasks or bottles of 75 ml and 200 ml respectively.All plants were grown in the fields of the Agricultural Research Institute (ARI), Tarnab Peshawar-Pakistan.

Host rearing
In order to get colonies of P. solenopsis from each of the five host plants, P. solenopsis was reared on the five aforementioned respective host plant species during four generations.For both field and laboratory experiments, gravid females of P. solenopsis from each of the five host plants, which were near to form nymphs sacs, were selected and individually put on 10-12 cm shoot with tender leaves on each of their respective host plant species and then covered with a very fine nylon cloth of mesh size 0.002 mm.After few days, the female started to produce young nymphs which were then transferred to their respective host plant species.Thereafter, when the nymphs reached to the adult stage they were transferred to new fresh shoots of their respective host plant species and left for giving young nymphs that will grow up to adults.For each host plant, this process was repeated to obtain 4 generations of 5 different P. solenopsis colonies.For field experiments, all colonies of P. solenopsis were reared directly on plants grown in the field while the colonies used for laboratory experiments were reared on leaves of their respective host plants in Petri dishes/plastic jars.

Parasitoids
The parasitoids were separately reared on each P. solenopsis colonies living separately on five host plant species.On each host plant, P. solenopsis adults which were near to form nymphs sacs were selected and covered by a very fine nylon cloth of mesh size 0.002 mm.After two days, P. solenopsis produced more than 50 nymphs at the same age.When the nymphs were fully spread on the shoots, the adults were removed.On each host plant, when most of the mealybug's nymphs reached the 3 rd instar then a pair of A. bambawalei was released inside the nylon bag for parasitization.After 24 hr, the parasitoid pair was removed and the nymphs were kept for mummy formation.This process was repeated to obtain 4 generations of 5 different A. bambawalei colonies on each P. solenopsis colony living separately on five host plant species.

No choice bioassays
In order to determine the percent of A. bambawalei parasitism under field conditions in no host-plant choice conditions, the upper shoots (12-15 cm) with tender leaves of each of the host plant were infested by 20 mealybugs of 2 nd instar, 3 rd instar, pre-ovipositing and adult females (a total of 80 mealybugs/host plant) and covered with nylon bag to avoid any nymphs/mealybug escape and to protect them from ants.These mealybugs were allowed to forage for 24 hrs to become fully established.Thereafter, one pair of freshly emerged A. bambawalei from the respective host plant was introduced and covered with nylon bag to protect them from other natural enemies' parasitism and predation.
The parasitoids were allowed to forage for parasitism for one week.Thereafter, the parasitoid pair was removed and transferred to another shoot which was previously infested by 20 mealybugs of each instar on the same pattern of 1 st week.After 2 nd week, the parasitoid pair was removed and placed in another new shoot during 3 rd week (if still alive) which was previously infested by the same pattern as during 1 st and 2 nd week.Weekly observations were made on pupae (mummies) formation and parasitoid emergence (P.solenopsis mummy that did not show emergence hole of an adult parasitoid during this time were noted as dead).Data on % parasitism, % emergence and sex ratio, female hind tibial length (mm) evaluated on 10 individuals and body weight (mg) evaluated on 5 individuals were recorded.This experiment was replicated ten times (n=10).

Host-plant choice test
In order to determine the percent of A. bambawalei parasitism under field condition in multiple host-plant choice conditions, for each host plant in small pots the upper shoots (12-15 cm) with tender leaves were infested randomly by 4 mealybugs each of 2 nd instar, 3 rd instar, preovipositing and adult females together (16 mealybugs/ plant i.e. a total of 80 mealybugs on all five host plants).Then all these pots were covered under a single nylon cloth to avoid any nymphs/mealybugs from escape and to protect them from ants.These mealybugs were allowed to forage for 24 hrs to become fully established.Thereafter, one pair of freshly emerged A. bambawalei was introduced.The parasitoids were allowed to forage for parasitism of mealybugs freely on all five host plants for one week.Thereafter, the parasitoid pair was removed and the shoots were covered separately by nylon bag and the same parasitoid pair was then transferred to other shoots, which were previously infested randomly by 80 mealybugs (16 individuals/host plant) of 2 nd instar, 3 rd instar, preovipositing and adult female.After one week, the pair of parasitoid was removed and if still alive then placed on other new shoots (for 3 rd week of parasitism) previously infested randomly by 80 mealybugs on the same pattern as during 1 st and 2 nd weeks.Weekly observations were made on pupae (mummies) formation and parasitoid emergence.Data on % parasitism, % emergence and sex ratio, female hind tibial length (mm) evaluated on 10 individuals and body weight (mg) evaluated on 5 individuals were recorded.This experiment was replicated ten times (n=10).

Parasitism under laboratory conditions
No host-plant choice test For each of host plants, the shoots fixed in glass conical flasks were infested by 20 mealybugs of 2 nd instar, 3 rd instar, pre-ovipositing and adult females (i.e. total of 80 mealybugs/host plant).When these mealybugs become fully established on each host plant then a pair of freshly/ young emerged A. bambawalei from the respective colony according to the host plant used was introduced and allowed to forage and parasitize the mealybugs for one week.After one week the parasitoid pair was removed and each shoot was covered again inside the nylon cloth.The same parasitoid pair was transferred to other conical flask (plant shoot) for another week of parasitism where each shoot was previously infested by 20 mealybugs of 2 nd instar, 3 rd instar, pre-ovipositing and adult female on the same pattern of 1 st week.The parasitoid pair was allowed to stay there for one week and then shifted (if still alive) to another conical flask with new shoot infested with 20 mealybugs of 2 nd instar, 3 rd instar, pre-ovipositing and adult female (i.e. total of 80 mealybugs/host plant) for 3 rd week of parasitism.After removing the parasitoid, the pupae (mealybugs mummies formed) were covered inside the nylon cloth till the adult emergence occurred.Data on % parasitism, % emergence and sex ratio, female hind tibial length (m) evaluated on 10 individuals and body weight (mg) evaluated on 5 individuals were recorded.This experiment was replicated ten times (n=10).

Host-plant choice test
For each of host plants, the shoots fixed in glass conical flasks were randomly infested by 16 mealybugs/ host plant) of 2 nd instar, 3 rd instar, pre-ovipositing and adult female together (i.e. a total of 80 mealybugs on all five host plants).Then a pair of freshly/young emerged A. bambawalei was introduced and allowed to forage and parasitize the mealybugs freely on all five host plants previously infested for one week.After one week the parasitoid pair was removed and each shoot was covered separately again inside the nylon cloth.The same parasitoid pair was transferred to other conical flask (plant shoot) for 2nd week of parasitism where these shoots were previously infested randomly by 16 mealybugs/host plant (a total of 80 mealybugs on all host plants) of the same mealybug instars.The parasitoid pair was allowed to stay there for one week and then shifted (if still alive) to another conical flask with new shoots previously infested by 16 mealybugs/host plant on the same pattern of 1 st and 2 nd week (a total of 80 mealybugs on all host plants) of the same instars as offered during 1 st and 2 nd week.After removing the parasitoid, the pupae (mealybug mummies formed) were covered inside the nylon cloth till the parasitoid emergence occurred.Data on % parasitism, % emergence and sex ratio, female hind tibial length (mm) evaluated on 10 individuals and body weight (mg) evaluated on 5 individuals were recorded.This experiment was replicated ten times (n=10).

Statistical analyses
All the data where needed were first transformed statistically and then subjected to ANOVA through a Completely Randomized Design (CRD).Untransformed results are presented in the tables.Means were separated by LSD at 5% level.Statistical tests were done using STATISTIX 8.1 package.

No choice bioassays
The results showed that the host plants influenced significantly the parasitism only at the third week of parasitism (Table I; F 4, 45 =0.32; p=0.8646 at the 1 st week; F 4, 45 =0.21; p=0.9313 at the 2 nd week and F 4, 45 =10.4; p=00001 at the 3 rd week).In fact, a negative correlation was observed between parasitism and the time passed for parasitism (r = -0.9693).However, the parasitism was significantly lower at the 3 rd week as compared to the previous weeks regardless of the host plant used.No difference was obtained for the % parasitoid emergence as well as for the sex ratio over the 3 weeks of parasitism (F 4, 45 =0.54; p=0.7078 for the 1 st week; F 4, 45 =0.51; p=0.7267 for the 2 nd week and F 4, 45 =0.06; p=0.994 for the 3 rd week; for sex ratio; F 4, 45 =0.07; p=0.9898).In contrast, the host plants influenced significantly the fitness of the parasitoid in terms of female hind tibial length and body weight.The female parasitoids coming from mealybugs reared on hibiscus exhibited a significantly longer hind tibia (F 4, 45 =6.31; p=0.0004) and body weight (F 4, 45 =3.95; p=0.0079) than the parasitoids coming from the mealybugs reared on the other plant species (Fig. 1). 1 Means in each column followed by the same letters are non significant at 5% level, using LSD test following ANOVA.   1 Means in each column followed by the same letters are non significant at 5% level, using LSD test following ANOVA.

Multiple choice bioassays
During multiple-choice bioassays, A. bambawalei females could chose its mealybug host in relation to the host plant on which it was feeding.In that case, A. bambawalei were more attracted to mealybugs living on hibiscus and okra showed highest parasitism for the 3 weeks of parasitism while the lowest was recorded on mealybugs living on eggplant (Table II; F 4, 45 =5.32; p=0.0013 for the 1 st week; F 4, 45 =3.9; p=0.0084 for the 2 nd week and F 4, 45 =3.45; p=0.0153 for the 3 rd week).No significant difference was obtained for % parasitoid emergence (F 4, 45 =0.68; p=0.6119 for the 1 st week; F 4, 45 =0.07; p=0.9906 for the 2 nd week and F 4, 45 =0.03; p=0.9978 for the 3 rd week).The sex ratio also did not differ significantly between hosts living on different plants (F 4, 45 =0.03; p=0.9986).In contrast, the host plants influenced significantly the fitness of the parasitoid in terms of hind tibial length and body weight.In a similar trend that the previous experiment under no choice conditions, the female parasitoids coming from mealybugs reared on hibiscus exhibited a significantly longer hind tibia (F 4, 45 =04.70; p=0.0030) and body weight (F 4, 45 =06.77; p=0.0002) than the parasitoids coming from the mealybugs reared on the other plant species (Fig. 2).

No choice bioassays
In a similar trend that the results obtained from the aforementioned field study under no choice conditions, the results showed that the host plant influenced significantly the parasitism only at the third week of parasitism (Table III, F 4, 45 =0.17; p=0.9533 at the 1 st week; F 4, 45 =0.89; p=0.4754 at the 2 nd week and F 4, 45 =5.36; p=0.0013 at the 3 rd week).However, the % parasitism was significantly lower at the 3 rd week as compared to the previous weeks regardless of the host plant used.No difference was obtained for the % parasitoid emergence as well as for the sex ratio over the 3 weeks of parasitism (for % parasitoid emergence: F 4, 45 =0.75; p=0.5606 for the 1 st week; F 4, 45 =0.18; p=0.9487 for the 2 nd week and F 4, 45 =0.28; p=0.8882 for the 3 rd week; for sex ratio: F 4, 45 =0.09; p=0.9836).In contrast, the host plants influenced significantly the fitness of the parasitoid in terms of hind tibial length and body weight.The female parasitoids coming from mealybugs reared on hibiscus exhibited a significantly longer hind tibia (F 4, 45 =07.36; p=0.0001) and body weight (F 4, 45 =07.23; p=0.0001) than the parasitoids coming from the mealybugs reared on the other plants (Fig. 3).

Multiple host-plant choice tests
In a similar trend that the results obtained from the aforementioned field study under multiple choice conditions, female's parasitoid Aenasius bambawalei can easily distinguish its host (mealybug) on different plants through movement and foraging behavior inside nylon cloth.A. bambawalei were more attracted to mealybugs feeding on hibiscus and okra showing highest % parasitism for the 3 weeks of parasitism while the lowest was recorded on mealybugs feeding on eggplant (Table IV;F=8.69;p=0.0001for the 1 st week; F 4, 45 =3.79; p=0.0097 for the 2 nd week and F=5.07; p=0.0018 for the 3 rd week).No significant difference was obtained for % parasitoid emergence (F 4, 45 =0.10 p=0.9829 for the 1 st week; F 4, 45 =0.32 p=0.8638 for the 2 nd week and F 4, 45 =0.06 p=0.9930 for the 3 rd week).The sex ratio did not differ also significantly between hosts feeding on different plants (F 4, 45 =0.04; p=0.9973).In contrast, the host plants influenced significantly the fitness of the parasitoid in terms of hind tibial length and body weight.In a similar trend that the previous experiment under no choice conditions, the female parasitoids coming from mealybugs reared on hibiscus exhibited a significantly longer hind tibia (F 4, 45 =3.64; p=0.0118) and body weight (F 4, 45 =03.34; p=0.0177) than the parasitoids coming from the mealybugs reared on the other plant species (Fig. 4).

Percent parasitism (weeks)
Percent parasitoid emergence (weeks) 1 Means in each column followed by the same letters are non significant at 5% level, using LSD test following ANOVA.   1 Means in each column followed by the same letters are non significant at 5% level, using LSD test following ANOVA.

DISCUSSION
Plant species not only influence phytophagous insect but also their associated natural enemies (Arif et al., 2012).For example, host plants structure and morphology can strongly influence the location and acceptance ability of parasitoids for their hosts (Wang et al., 2013;Wang et al., 2009).In our study, the host plant on which P. solenopsis was feeding on, was significantly influencing the host acceptance by A. bambawalei in terms of parasitism.The plant used in this study exhibited very different morphology characteristics, like leaf structure and shape, number of trichomes per centimeter surface area and leaf thickness.Among the plant species used in our study, hibiscus exhibits very thin leaves and less number of trichomes as compared to the other plant species, probably helping the wasp to encounter more easily its host.
Moreover, plants provide generally chemical signals used as foraging cues by parasitic and predaceous arthropods (Godfray, 1994;Turlings et al., 1995).Apart from pheromones, the chemical compounds originating from herbivores are at most slightly volatile and can only be detected at close range (Vet and Dicke, 1992).Plant volatiles released in response to mechanical damage by herbivores, including green-leaf volatiles and constitutive secondary compounds, are known to be attractive to various parasitoids (Steinberg et al., 1993;Mattiaci et al., 1994).Volatiles released in response to herbivore feeding are generally reliable indicators of herbivore presence and can potentially bring parasitoids in close proximity to their hosts.In the present study, we observed that during multiple-choice conditions the female parasitoid A. bambawalei could easily make a choice of its host according to the plant species on which it was feeding.These preferences for mealybugs feeding on hibiscus can be therefore attributed also to chemical cues more attractive to A. bambawalei than the chemicals emitted by the other infested plant species.This corroborated the variability of this parasitoid species efficiency according to the locality and the plants on which P. solenopsis is feeding observed by Arif et al. (2012) in Pakistan.
We recorded also in our study a higher number of P. solenopsis parasitized during the 1 st week of parasitism, indicating a clear negative correlation between the parasitism and the age of the parasitoid.Ashfaq et al. (2010) reported that in case of A. bambawalei parasitism efficacy, a significant reduction in parasitism with an increase in the age of parasitoid was observed.Such observation were also reported by Bertschy et al. (2000) for another parasitoid species Aenasius vexans (Kerrich) that after emergence during the first 5 days this parasitoid had parasitized the greatest number of mealybug and after that the parasitizing efficacy of the A. vexans decreased drastically.
However, the differences of parasitism between plant species were only highlighted at the 3 rd week of parasitism not before.The highest % of parasitism on hibiscus and okra particularly in 3rd week also suggested that the parasitoids on these plants can live long and also get more energy or nutritional needs for a better rate of parasitism on such plant species.It is well reported in the literature that plants supply direct resources for parasitoids and predators for their survival, fecundity, and the efficiency of parasitism and predation (Sirot and Bernstein, 1996;Consuelo et al., 2000).Moreover, it is observed that the absence or the incompatibility of direct food sources from plant can result in a reduced parasitizing efficiency by the parasitoids, spending more time to search for food first than for hosts searching and parasitism (Lewis and Tumlinson, 1998).
Some herbivores can use plant secondary chemicals in defense against their parasitoids, which ultimately caused the parasitoid encapsulation (Singer, 2001;Singer et al., 2004).Blumberg (1995) reported that a high incidence of encapsulation may also cause difficulties in mass rearing of parasitoids.Our results suggest that in the case of A. bambawalei larval encapsulation was not probably occurring since adult emergence rates from all mealybugs feeding on these five host plants species remained non significant.
Moreover, it is well documented in the literature that in most hymenopterous parasitoids the accurate assessment of the host suitability for offspring development is important since females are supposed to maximize their reproductive success by ovipositing on high-quality hosts and that the plants influence strongly through the hosts the development and performance of the third trophic level (Godfray, 1994).However, little is known on the influence of the host plant on sex allocation process of the parasitic wasps (Ode, 2006).In our study, it was clearly showed that the host plant species did not influence the sex ratio.This is in agreement with the works of Ashfaq et al. (2010), Arif et al. (2012) and Fand et al. (2011).In contrast, our study showed that the host plant species influenced significantly the fitness of A. bambawalei females expressed in terms of tibia length and body weight.The female wasps grew better on mealybugs fed on hibiscus and okra than on the hosts fed on the other plant species used in this study.These results are in parallel with Ode (2006) who reported that the host plant affects the parasitoid fitness by a variety of means, including survivorship, clutch size, body size, and/or fecundity and negative impacts on parasitoid fitness may occur when the developing parasitoid encounters the plant toxin in the hemolymph or tissue of its herbivorous host.Similarly, Ode (2006) and Mooney et al. (2012) reported that host quality and chemistry could influence the parasitoid performance, either directly through plant toxins or metabolites in the host's hemolymph or tissues, or indirectly via changes in host quality or size.Moreover, similarly Yang and Sadof (1995) reported that Coleus sp leaf variegation can affect the rate of fecundity, survivorship, and population growth of both the citrus mealybug, Planococcus citri (Risso) and its associated parasitoid Leptomastix dactylopii (Howard).In the same way, in another study, it was reported that the host quality or size is commonly correlated to the parasitoid fitness, especially in idiobiont parasitoids, whose offspring may depend on the quality and size of the host at the time of oviposition (Gols and Harvey, 2008;Fortuna et al., 2012).

CONCLUSION
The current study revealed a significant influence of the host plant species mostly on the % parasitism and the parasitoid fitness (expressed here in term of tibia length and body weight) where hibiscus was the most suitable host plant.The originality of this study was to show this using colonies of both host and parasitoid previously reared during 4 generations on each of the five plant species tested in this study.All these aspects explain well why the efficiency of this parasitoid is variable according to the locality and the plants on which P. solenopsis is feeding.In addition, this study allows us to recommend the use of hibiscus, Hibiscus rosa sinensis, in the mass rearing process of A. bambawalei for a biological control program to control P. solenopsis.

Fig. 1 .
Fig. 1.Influence of P. solenopsis host plants on the fitness of Aenasius bambawalei under field conditions (no choice bioassays).Bars topped up by different litters within each parameter are significantly different at 5% level of probability.

Fig. 2 .
Fig. 2. Influence of P. solenopsis host plants on the fitness of Aenasius bambawalei under field conditions (choice bioassays).Bars topped up by different litters within each parameter are significantly different at 5% level of probability.

Fig. 3 .
Fig. 3. Influence of P. solenopsis host plants on the fitness of Aenasius bambawalei under laboratory conditions (no choice bioassays).Bars topped up by different litters within each parameter are significantly different at 5% level of probability.

Fig. 4 .
Fig. 4. Influence of P. solenopsis host plants on the fitness of Aenasius bambawalei under laboratory conditions (choice bioassays).Bars topped up by different litters within each parameter are significantly different at 5% level of probability.